Shirley Zhu/ Genomic DNA labeling
Array-Based Comparative Genomic Hybridization Protocol
(Based on the protocol from Pollack lab)
Digestion and Purification of Genomic DNA
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4 ug of DNA from sample (adjust volume accordingly for each sample), add 1 ul
of DpnII, 4ul of 10X DpnII buffer, up to 40ul of total volume. (Water and DNA
together should be 35 l; then heat it at 60/55C for 10 min; then add DpnII
and the buffer. This helps dissolve DNA in water well.) Mix well.
Digest for 1.5 hours at 37 C
Inactivate by heating at 65 C for 20 min, then snap cool on ice Add 60ul of TE
(ph8.0) to each sample to bring volume up to 100ul
Add 500ul (5 volume) of Buffer PB (from QIAGEN, QIAquick PCR Purification
Kit, CAT#28194), and invert several times before a quick spin
Apply 600ul to column, spin for 30 seconds, discard flow-thru
Add 750ul of Buffer PE and spin for 30 second
Spin additional 1 min to dry column
Place column into a new eppendorf tube
Add 23ul of TE (ph8.0) directly on top and center of the filter
Let it sit at RT for 1 min
Spin 1 min at max. RCF to recover bound DNA
Labeling of Genomic DNA Fragments
-Transfer the 21-23ul of DNA to a micro centrifuge tube
-Add 20ul of 2.5 X random prime mix( Invitrogen, BioPrime DNA Labeling System Kit,
CAT# 18094-011) to 21-23ul of DNA
-Boil 5 min, then place on ice for 5 min, do a quick spin at 4 C
-Mixture: DNA
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41ul
10X dCTPdNTP mix
5ul
Cy3/Cy5
3ul
Klenow
1ul (from the Kit)
Total volume
50 ul
Tap gently and give it a quick spin
Incubate 2 hours at 37C
Add 5 ul of stop solution( from the Kit)and put back on ice.
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Shirley Zhu/ Genomic DNA labeling
Concentrating Labeling Probes and hybridization to arrays
-Combine Cy3 and Cy5, put Cy3 to Cy5
-Add 400 ul of TE (pH8.0) to each combined probe
-Apply the labeled probe to a Microcon YM30 (Millipore, Cat. No.: 42410)
-Spin at 12,000xg for 11 min, make the volume less than 20 ul or so(½ of the filter is
visible),
-Discard flow-thru, wash with additional 500 ul TE (pH8.0), and spin at 12,000xg for 11
min, make the volume less than 20 ul
-Make mix:
Human Cot-1 DNA
50 ul
Yeast tRNA
20 ul( 5ug/ul)
Poly dT-dA
4 ul(5ug/ul)
TE (pH8.0)
450ul
Total
about 520 ul
-Mix well, add to the filter
-Spin for 11-12 min at 12,000xg, make the volume less than 32 ul
-Invert the filter to a new tube, spin 2 min to recover the labeled probe
-Adjust the volume by Dnase free water to 32 ul
-Then make:
Probe
32 ul
20X SSC
6.8 ul
10 % SDS
1.2 ul
Total volume
40 ul
( wipe off additional SDS on pipette tip with side of gloves)
-Mix lightly to minimize bubbles, and give a quick spin
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Shirley Zhu/ Genomic DNA labeling
-Denature probe(s) by heating for 2 min at 100 C, then quick spin
-Incubate at 37C for 30 min for Cot-1 to block repetitive DNA in sample
-Pre-warm Hyb charmbers on top of 65 C Hyb water bath
-Spin DNA probe 5 min at max rcf
-Working quickly , place 40 ul of probe onto array
-Add 20 ul of 3X SSC drops scattered evenly across the top of the cover slip. Tighten
screws and incubate at 65 C overnight.
Wash arrays as with mRNA labeling protocol and scan:
-Then transfer the slides to 2X SSC/0.03%SDS at 65C, shake the slide holder up and
down vigorously for 5 min, making sure slides never out solution.
-Wash slides in 2XSSC for 5 min, same as above or with a shaker
-Wash slides in 1XSSC for 5 min, same as above
-Wash slides in 0.2XSSC for 5 min, twice
-Spin slides down in centrifuge at 500 rpm for 5 min (prepare everything in advance, so
transfer of slide rack to holder is very quickly done)
- Clean box needed to transport slides to scanner
-Scan immediately!
Washing buffer:
20X SSC stock solution
500 ml of 2X SSC/0.03% SDS
500 ml of 2X SSC
500 ml of 1X SSC
500 ml of 0.2 X SSC
50 ml
50 ml
25 ml
5 ml
10% SDS
1.5 ml at 65 C
Note:
The first washing step should be performed at 65° C; this appears to significantly
increase the specific to non-specific hybridization signal.
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